The development of the mass spectrometric methodology for the identification of covalently bound adducts to hemoglobin and other circulating blood proteins, such as serum albumin, will be continued with the aim to detect such adducts at an abundance level of 1:10(5) (adducted hemoglobin to normal hemoglobin). The present approach of searching for adducts onto cysteine 93 or 112 of the human beta-chain will be continued using chemical cleavage at the unreacted cysteines, enriching the uncleaved reacted material and analysis of the tryptic peptides by mass spectrometry. Vinylpyridine adducts will be the model. When the point is reached where one adduct in 10(5) hemoglobin molecules can be reliably identified, the methodology will be used to detect this adduct in smokers because vinylpyridine is a component of tobacco smoke. In parallel, an entirely different approach will be developed that should be more generally applicable and also detect adducts at other amino acids, such as histidine, which can undergo nucleophilic reactions and therefor pick up potential nucleophiles. Further digestion of a tryptic (or other specific) digest with appropriate exopeptidases (for example, carboxypeptidase P) reduces a protein such as hemoglobin to its constituent amino acids. However, it has been found that if an amino acid is modified with a large substituent, the action of the exopeptidase stops at that point. Thus there will be a peptide of a certain (predictable) size left for each modified amino acid. The pool of free amino acids can be removed by high performance liquid chromatography (HPLC) and the remaining peptide(s) detected by rechromatographing the remainder. The peptide and at least the major structural features of the adduct will then be identified by mass spectrometry.